CN117347626B - Human insulin-like growth factor binding protein-1 chemiluminescence immunoassay kit and preparation method thereof - Google Patents
Human insulin-like growth factor binding protein-1 chemiluminescence immunoassay kit and preparation method thereof Download PDFInfo
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- 238000003018 immunoassay Methods 0.000 title claims abstract description 31
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
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- 102000004375 Insulin-like growth factor-binding protein 1 Human genes 0.000 claims abstract description 39
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 9
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 claims description 8
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4745—Insulin-like growth factor binding protein
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2470/00—Immunochemical assays or immunoassays characterised by the reaction format or reaction type
- G01N2470/04—Sandwich assay format
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
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- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a human insulin-like growth factor binding protein-1 chemiluminescence immunoassay kit, which comprises a first reagent and a second reagent, wherein the first reagent is a streptavidin magnetic microsphere and biotinylated IGFBP-1 antibody solution, and the second reagent is an IGFBP-1 antibody marked by alkaline phosphatase; and a sample processing liquid. Also discloses a preparation method of the human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit. The invention adopts streptavidin magnetic microsphere as carrier, biotin marks one IGFBP-1 antibody, alkaline phosphatase marks the other IGFBP-1 antibody, and the chemiluminescence system of the other IGFBP-1 antibody quantitatively detects IGFBP-1 in cervical secretion or vaginal discharge, and can be matched with a large-scale chemiluminescent immunoassay analyzer, a small-scale chemiluminescent immunoassay analyzer and a single chemiluminescent immunoassay analyzer for use, thereby meeting different clinical requirements.
Description
Technical Field
The invention relates to the technical field of biological protein detection, in particular to a chemiluminescent immunoassay kit for human insulin-like growth factor binding protein-1 and a preparation method thereof.
Background
The family of Insulin Growth Factor Binding Proteins (IGFBP) is a group of soluble proteins that specifically bind and regulate insulin growth factors I, II (IGF-I, II), including 6 sequentially discovered IGFBP's, designated IGFBP1-6, respectively. IGFBP-1 consists of 234 amino acids and has a molecular weight of about 25KD. IGFBP-1 in the blood circulation is produced primarily by hepatocytes and plays an important role in the female reproductive system as an autocrine and paracrine factor, particularly for ovulation, implantation of fertilized eggs to fetal delivery. IGFBP-1 in amniotic fluid is synthesized and secreted by the maternal liver, decidua and fetus. The embryo ectocoelom in early gestation has very high IGFBP-1, little amniotic fluid, fusion of amniotic membrane and chorion/decidua in mid gestation, and the IGFBP-1 concentration in amniotic fluid is increased by several orders of magnitude and 100-1000 times higher than that of maternal blood, and is not influenced by cervical mucus, urine and semen. IGFBP-1 is not normally detected in cervical secretions after 12 weeks gestation, but is released into cervical secretions when the decidua separates from the chorion prior to premature delivery. Thus, premature delivery can be predicted by measurement of IGFBP-1. Premature rupture of the fetal membranes is one of the major causes of morbidity and mortality in perinatal females, with a incidence of about 10-17%. When the fetal membranes are early broken, IGFBP-1 in amniotic fluid leaks into cervical mucus, so that detection of IGFBP-1 in vaginal or cervical secretion can assist diagnosis of the fetal membranes, and the sensitivity and specificity of the detection are better than those of the traditional diagnosis indexes.
The invention patent 'an insulin-like growth factor binding protein-1 detection kit' declared by Chongqing Qiande biotechnology Co-Ltd adopts a quantitative detection method of a detection test paper card of a nitrocellulose membrane coated with a detection antibody and a fluorescent microsphere marked antibody; the invention patent 'detection system and reagent based on up-conversion luminescence human insulin-like growth factor binding protein-1 (IGFBP-1) chip' filed by Shanghai Kai JING biotechnology Co Ltd adopts up-conversion luminescence UCP particle labeled antibody, and a sample is manually dripped into a detection cup for reaction and then is inserted into a fluorescence detector for detection. The colloidal gold/quantum dot fluorescent detection has the problems of insufficient accuracy, repeatability and sensitivity, and detection errors are caused by manual operation, so that a novel human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit and a preparation method thereof are needed to solve the problems.
Disclosure of Invention
The invention aims to solve the technical problem of providing a human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit and a preparation method thereof, wherein a streptavidin magnetic microsphere is adopted as a carrier, biotin is used for marking one strain of IGFBP-1 antibody, alkaline phosphatase is used for marking a chemiluminescent system of the other strain of IGFBP-1 antibody to quantitatively detect IGFBP-1 in cervical secretions or vaginal effluents, and the kit can be matched with a large chemiluminescent immunoassay analyzer, a small chemiluminescent immunoassay analyzer and a single chemiluminescent immunoassay analyzer for use.
In order to solve the technical problems, the invention adopts a technical scheme that: a human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit is provided, comprising the following reagents:
the first reagent is a solution of streptavidin magnetic microspheres and biotinylated IGFBP-1 antibody; and
a second reagent which is an IGFBP-1 antibody labeled by alkaline phosphatase; and
sample treatment fluid.
In a preferred embodiment of the present invention, the streptavidin magnetic microsphere is prepared by covalently coupling activated carboxyl magnetic microsphere with streptavidin.
In a preferred embodiment of the invention, the concentration of the first reagent is from 50ug/mL to 300ug/mL.
In a preferred embodiment of the invention, the concentration of the second agent is between 0.05ug/ml and 0.200ug/ml.
In a preferred embodiment of the present invention, the sample processing liquid comprises the following components in mass fraction:
matrix buffer 0.02-0.1M/L
NaCl 0.9%
Matrix metalloproteinase inhibitor 10.0-50.0mM/mL
Brij-35 0.5%
0.05 to 0.3 percent of polyvinylpyrrolidone
Sodium dodecyl sulfate 0.01-0.025%
Trehalose 0.05% -0.5%
BSA 0.1%-0.5%
Dithiothreitol 0.005-0.03%
Sodium bisulphite 0.02-0.1%
0.02% -0.05% of chlorhexidine acetate.
In order to solve the technical problems, the invention adopts another technical scheme that: the preparation method of the human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit comprises the following steps:
s1: preparation of the first reagent:
s101: carboxyl magnetic microsphere activation: taking the suspension magnetic microsphere liquid into a centrifuge tube; removing the supernatant, adding 0.1M MES, suspending the magnetic microspheres, and repeatedly cleaning the magnetic microspheres for 3-5 times; adding EDC solution and N-hydroxysuccinimide solution to activate the magnetic microspheres, and repeatedly cleaning the magnetic microspheres for 2-5 times;
s102: covalent coupling of activated magnetic microspheres with streptavidin: preparing a reaction solution, adding activated carboxyl magnetic microspheres into the reaction solution, fully and uniformly mixing the suspension magnetic microspheres for reaction, removing supernatant in a centrifuge tube, adding 0.1M MES, suspending the magnetic microspheres, and repeatedly cleaning the magnetic microspheres for 3-5 times; adding the reaction solution into the washed magnetic microspheres, fully and uniformly mixing to suspend the magnetic microspheres for reaction, removing supernatant in a centrifuge tube, washing the magnetic microspheres with TBST solution, and repeating the washing for 3-5 times;
s103: biotinylated antibody: preparing a Biotin labeling reagent NHS-Biotin, placing the NHS-Biotin into a centrifuge tube, adding IGFBP-1 antibody purified by PBS buffer solution, fully blowing and uniformly mixing, standing at 37 ℃ in a dark place for 15min, and incubating at room temperature in a dark place for 45min in a rotating way; transferring the reacted biotinylated antibody into a dialysis bag for dialysis, and transferring into a centrifuge tube;
s104: the streptavidin magnetic microsphere is connected with a biotinylation antibody: preparing a connection reaction solution; placing streptavidin magnetic microspheres on a magnetic separation frame, and removing the supernatant; adding the connection reaction liquid, placing the connection reaction liquid on a magnetic separation frame, removing the supernatant, and repeatedly cleaning for 2-3 times; suspending streptavidin magnetic microspheres by using the connection reaction liquid, adding a biotinylation antibody, supplementing the reaction volume by using the connection reaction liquid, reacting, removing supernatant in a centrifuge tube, and repeatedly cleaning for 3-5 times;
s2: preparation of the second reagent:
s201: antibody treatment: adding the antibody with the adjusted concentration into SATA solution, performing light-proof rotary reaction at room temperature, and purifying and concentrating with PBS buffer solution for 5-8 times after the reaction is finished; collecting antibody, adding hydroxylamine hydrochloride solution, reacting at room temperature, purifying and concentrating with PBS buffer solution for 5-8 times after the reaction is finished;
s202: alkaline phosphatase activation: weighing SMCC, dissolving in DMSO, and preparing into SMCC; adding the SMCC solution into the PBS solution to obtain the SMCC solution; adding an ALP solution into an SMCC solution, and reacting at room temperature; dialyzing with PBS buffer solution after the reaction is finished;
s203: marking: collecting ALP activating enzyme after dialysis and purification, adding the treated antibody, supplementing the volume with PBS buffer solution, and reacting at room temperature in a dark place; dialyzing with PBS buffer solution after the reaction is finished; collecting the antibody after dialysis and purification, and diluting to the use concentration by using a marking buffer solution;
s3: sample treatment liquid was prepared.
In a preferred embodiment of the invention, 0.15mg of streptavidin is dissolved in 1.35mL of 0.1M MES solution, the pH of the MES solution is 4.0-7.0, and then 150ul of 1-hydroxybenzotriazole dissolved in tetrahydrofuran and 10mg/mL of 1-hydroxybenzotriazole are added and thoroughly mixed.
In a preferred embodiment of the invention, 0.05g of polyethylene glycol and 0.09g of NaCl are weighed and dissolved in 10mL of 10mM PBS buffer solution, so that the pH of the connecting reaction solution is 8.0-8.5.
The beneficial effects of the invention are as follows:
(1) The invention adopts streptavidin magnetic microsphere as carrier, biotin marks one IGFBP-1 antibody, alkaline phosphatase marks the other IGFBP-1 antibody, and the double antibody sandwich method carries out quantitative detection on IGFBP-1 in cervical secretion or vaginal discharge, and the performances of sensitivity, repeatability and the like are superior to those of immunochromatography, manual operation and the like;
(2) According to the invention, each component of the reagent is packaged into a specific reagent strip or a reagent bottle, so that the reagent can be matched with a single chemiluminescent immunoassay analyzer, a small chemiluminescent immunoassay analyzer and a large chemiluminescent immunoassay analyzer for use, different clinical requirements are met, the problems of insufficient accuracy, repeatability and sensitivity of the conventional colloidal gold/quantum dot fluorescent detection are solved, full-automatic operation is realized, errors caused by manual operation are avoided, and quantitative detection of IGFBP-1 in cervical secretion or vaginal discharge is realized.
Drawings
FIG. 1 is a schematic representation of the dose-response curves of a kit of the invention for zero concentration standard and five concentration standard lot 1;
FIG. 2 is a schematic representation of the dose-response curves for a kit of the invention for zero concentration standard and five concentration standard lot 2;
FIG. 3 is a schematic representation of the dose-response curves for a kit of the invention for zero concentration standard and five concentration standard lot 3;
FIG. 4 is a schematic diagram of a correlation comparison of clinical samples using the development reagent and the contrast reagent (colloidal gold) of the present invention.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making clear and defining the scope of the present invention.
A human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit for quantitative detection of IGFBP-1 in cervical secretions or vaginal effluents comprising a detection reagent comprising a first reagent, a second reagent, and a sample processing fluid. The first reagent is a solution of streptavidin magnetic microspheres and biotinylated IGFBP-1 antibody, and the second reagent is an IGFBP-1 antibody labeled by alkaline phosphatase.
Preferably, the streptavidin magnetic microsphere is prepared by covalent coupling of activated carboxyl magnetic microsphere and streptavidin. Biotin labeling the antibodies were labeled with biotin by a coupling method.
The sample treatment fluid comprises the following components in mass fraction:
matrix buffer 0.02-0.1M/L
NaCl 0.9%
Matrix metalloproteinase inhibitor 10.0-50.0mM/mL
Brij-35 0.5%
0.05 to 0.3 percent of polyvinylpyrrolidone
Sodium dodecyl sulfate 0.01-0.025%
Trehalose 0.05% -0.5%
BSA 0.1%-0.5%
Dithiothreitol 0.005-0.03%
Sodium bisulphite 0.02-0.1%
0.02% -0.05% of chlorhexidine acetate.
Wherein the substrate buffer solution is PB solution with pH of 7.5-8.5 and concentration of 0.02-0.1M/L.
The collected sample is treated by adopting self-made sample treatment liquid, so that the collected cervical secretion or vaginal effluent is completely eluted, and IGFBP-1 can be stably stored for 48 hours at the temperature of 2-8 ℃ in the sample treatment liquid.
The IGFBP-1 antibody may be a monoclonal antibody or a polyclonal antibody, and is not limited thereto.
The invention also provides a preparation method of the human insulin-like growth factor binding protein-1 chemiluminescence immunoassay kit, which comprises the following steps:
s1: preparation of the first reagent:
s101: carboxyl magnetic microsphere activation:
(1) Fully suspending the magnetic microspheres by using a vortex mixer, and taking 5.0-20.0 mg of suspension magnetic microsphere liquid into a centrifuge tube;
(2) Place the centrifuge tube on a magnetic separation rack for about 1 minute, carefully remove the supernatant; 1mL of 0.1M MES (pH 4.0-7.0) was added to suspend the magnetic microspheres; then place on the magnetic separation rack for about 1 minute, carefully remove the supernatant (repeatedly wash the magnetic microspheres 3-5 times);
(3) Adding 5 mu L-120 mu L of 10mg/mL EDC solution and 5 mu L-120 mu L of 10mg/mL N-hydroxysuccinimide (NHS) solution, and fully mixing by a vortex mixer to activate the magnetic microspheres at room temperature for 30min;
(4) And (3) placing the activated magnetic microspheres on a magnetic separation frame, and repeatedly cleaning the magnetic microspheres for 2-5 times according to the method of the step (2).
S102: covalent coupling of activated magnetic microspheres with streptavidin:
(1) The reaction solution: 0.15mg of streptavidin was dissolved in 1.35mL of a 0.1M MES (pH 4.0-7.0) solution, and 150ul of 1-hydroxybenzotriazole (HOBt) dissolved in tetrahydrofuran (HOBt concentration: 10 mg/mL) was added thereto, followed by thoroughly mixing.
(2) Adding 5.0mg-20.0mg of activated carboxyl magnetic microspheres into 0.5mL-1.0mL of reaction liquid, fully and uniformly mixing the suspension magnetic microspheres at room temperature by using a vortex mixer to react for 2 hours, and fully and uniformly mixing the suspension magnetic microspheres at the temperature of 2-8 ℃ to react for 5-8 hours.
(3) Place the centrifuge tube on a magnetic separation rack for about 1 minute, carefully remove the supernatant; the magnetic microspheres were suspended by thoroughly mixing with a 0.1M MES (pH 4.0-7.0) solution, with a vortex mixer, placed on a magnetic separation rack for about 1 minute, and the supernatant carefully removed (the magnetic microspheres were repeatedly washed 3-5 times).
(4) Adding 0.2-0.5 mL of reaction solution into the washed magnetic microspheres, and fully and uniformly mixing the suspension magnetic microspheres by using a vortex mixer at 2-8 ℃ for reaction for 2-3 hours.
(5) Place the centrifuge tube on a magnetic separation rack for about 1 minute, carefully remove the supernatant; the magnetic microspheres were washed with 1mL TBST solution and repeated 3-5 times. Diluting to 20mg/mL with TBST solution, and storing at 2-8deg.C for use.
S103: biotinylated antibody:
(1) Biotin: 1.0mg of NHS-Biotin was weighed and dissolved in 100. Mu.L of LDMSO, and 50ul of the dissolved NHS-Biotin was added to 950. Mu.L of 10mM PBS buffer (pH 7.0-8.0) to prepare 0.5mg/mL of NHS-Biotin.
(2) mu.L-100. Mu.L of 0.5mg/mL NHS-Biotin was placed in a 1.5mL centrifuge tube, and 0.1mg of IGFBP-1 antibody purified by 10mM PBS buffer (pH 7.0-8.0) was added thereto, followed by thoroughly pipetting and mixing. After being kept stand for 15min at 37 ℃ in a dark place, the culture medium is rotated and incubated for 45min at room temperature in a dark place.
(3) The reacted biotinylated antibody was transferred to a dialysis bag, dialyzed against 10mM PBS buffer (pH 7.0-8.0) for 6-8 hours, and the buffer was changed once. The dialyzed biotinylated antibody was transferred to a centrifuge tube.
S104: the streptavidin magnetic microsphere is connected with a biotinylation antibody:
(1) And (3) connecting the reaction liquid: 0.05g of polyethylene glycol, 0.09g of NaCl are weighed and dissolved in 10mL of 10mM PBS buffer, and the final pH is 8.0-8.5.
(2) Taking 20mg/mL streptavidin magnetic microspheres on a magnetic separation rack for about 1 minute, carefully removing the supernatant; 1mL of the ligation reaction was added and the mixture was placed on a magnetic separation rack for about 1 minute, and the supernatant was carefully removed (2-3 washes repeated).
(3) The streptavidin magnetic microspheres were suspended in 0.5mL of ligation reaction, 0.1mg of biotinylated antibody was added, and the reaction volume was made up to 1.0mL with ligation reaction. Placing the mixture at 2-8 ℃ for reaction for 1-2h.
(4) Place the centrifuge tube on a magnetic separation rack for about 1 minute, carefully remove the supernatant; the washing was repeated 3-5 times with 1mL TBST solution. Diluting the magnetic microsphere with TBST solution to the use concentration, and storing at 2-8deg.C for use.
S2: preparation of the second reagent:
s201: antibody treatment:
adjusting the concentration of the antibody to 5.0mg/mL by using 0.02-0.10M PBS (pH 6.50-8.00), taking 50.0-100.0 mu L of the antibody of 5.0mg/mL, adding 50.0-100.0 mu L of 0.1-0.6mg/mL SATA solution, and rotating at room temperature in a dark place for reaction for 20-60min. After the reaction, the mixture is purified and concentrated for 5 to 8 times by using a 50kD ultrafiltration centrifuge tube and 0.02 to 0.10M PBS (pH 6.50 to 8.00) buffer solution. About 100. Mu.L of antibody was collected, and 50-100. Mu.L of a 0.1-0.5M hydroxylamine hydrochloride solution was added thereto to react at room temperature for 1-3 hours. After the reaction, the mixture is purified and concentrated for 5 to 8 times by using a 50kD ultrafiltration centrifuge tube and 0.02 to 0.10M PBS (pH 6.50 to 8.00) buffer solution. Approximately 100. Mu.L of antibody was collected.
S202: alkaline phosphatase activation:
weighing 2.0-8.0mg of SMCC, dissolving in 1.0mL of DMSO, and preparing into 2.0-8.0mg/mL of SMCC; 100. Mu.L of 2.0-8.0mg/mL SMCC solution was added to 0.9mL of 0.1M PBS (pH 6.50-8.00) to give 0.2-0.8 mg/mL SMCC solution. Taking 10-50 mu L of ALP solution with the concentration of 10.0-40.0mg/mL, adding 50.0-150.0 mu L of SMCC solution with the concentration of 0.2-0.8 mg/mL, and reacting for 40-90min at room temperature. After the reaction, the mixture was dialyzed against 0.01-0.02M PBS (pH 6.50-8.00) for 6-8 hours.
S203: marking:
the ALP activating enzyme after dialysis purification is collected, the treated antibody is added, the buffer solution with 0.1M PBS (pH 6.50-8.00) is used for supplementing 1mL, and the reaction is carried out at room temperature for 4-8h in a dark place. After the reaction, the mixture was dialyzed against 0.01-0.02M PBS (pH 6.50-8.00) for 8-12h. The antibody after dialysis purification is collected, diluted to the use concentration by a labeling buffer solution and stored at 2-8 ℃ for standby.
S3: sample treatment liquid was prepared.
The sample processing liquid is prepared as described above, and will not be described here again.
The detection method of the human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit comprises the following steps:
the first step: inserting the sampled sampling cotton swab into a centrifuge tube with sample treatment fluid, breaking the cotton swab, covering the tube cover of the centrifuge tube tightly, placing the centrifuge tube in an oscillator to oscillate for 1-3min, and fully eluting secretion or effluent to obtain a qualified sample.
And a second step of: uniformly mixing 10ul-30ul of a sample to be tested (a sample treated by a sample treatment solution), 20ul-50ul of a magnetic microsphere-biotin labeled IGFBP-1 antibody solution (the concentration is 50ug/mL-300 ug/mL) and 10ul-30ul of an alkaline phosphatase labeled IGFBP-1 antibody solution (the concentration is 0.05ug/mL-0.200 ug/mL) at 37 ℃ for reaction for 10min;
and a third step of: 200ul of washing liquid is sucked, and the washing is repeated for 3 times;
fourth step: adding 200ul of substrate liquid into the washed reaction cup/hole, reacting for 3-5min, and automatically measuring the luminescence value by an instrument;
fifth step: the concentration of IGFBP-1 in the sample is calculated by substituting the resulting luminescence value into a standard curve equation using a calibration curve of known concentration.
The matched detector comprises a large-scale chemiluminescent immunoassay analyzer, a small-scale chemiluminescent immunoassay analyzer and a single chemiluminescent immunoassay analyzer.
Specifically, the principle of the assay method is a double-antibody sandwich enzymatic chemiluminescence method.
Three batches of the kit provided by the invention were selected for performance testing.
1. Linearity and sensitivity
The zero concentration standard (S0) solution is provided with 20 compound wells, and the other 5 concentration standards S1-S5 are provided with single wells. Drawing a standard curve according to the theoretical concentration and the luminescence value of the standard substance to obtain a kit dose-response curve, wherein the results are shown in fig. 1, 2 and 3, and three batches of R2 are respectively as follows: 0.9994, 0.9991, 0.9987. Calculating the average value (Mean) and Standard Deviation (SD) of 20S 0 luminescence values, and calculating the concentration value of mean+2SD according to a fitting equation, namely the sensitivity of the kit, wherein the detection result of the sensitivity of the kit is shown in Table 1, and the sensitivity of the three batches of kits is not higher than: 0.24ng/ml.
Samples of high (120.00 ng/ml) and low (25.00 ng/ml) concentrations were taken and each sample was repeatedly tested 10 times using three separate kits. The kit reproducibility test results (table 2) show the intra-kit lot differences: < 3.00%, difference between batches: less than 3.10 percent.
3. Specificity (specificity)
To samples of high (120.00 ng/ml) and low (25.00 ng/ml) concentrations, hemoglobin of 5.0mg/ml, 15.0mg/ml and leukocyte esterase of 10U/ml and 200U/ml were added, respectively, to obtain samples to be tested. Each sample was tested with lot 1 reagent and the mean of the measurements should be within the mean target concentration (M) ±2 Standard Deviation (SD).
The results of the high concentration sample specificity experiments are shown in Table 3, and the results of the low concentration sample specificity experiments are shown in Table 4: and adding hemoglobin and leukocyte esterase with different concentrations into the high-concentration sample and the low-concentration sample respectively, wherein the average value of detection results is in the target concentration range of 111.77 ng/ml-115.97 ng/ml and 20.50 ng/ml-21.58 ng/ml.
4. Accuracy of
The calibration material of the kit (concentration value: school 1:2.00ng/ml; school 2:20.00ng/ml; school 3:100.00 ng/ml) was used as a sample, and each repeated measurement was carried out 3 times, the test result was recorded as (Xi), and the relative deviation (Bi) was calculated according to the formula (1) and was within + -10%. The results showed that the relative deviation was within + -10% (the accuracy test results are shown in Table 5).
Bi=（Xi-T）/T×100% （1）
Note that: wherein Bi-relative deviation; xi-measuring concentration; t-calibration concentration.
5. Clinical sample correlation detection
A total of 33 clinical samples were collected and compared with the development reagent and the comparison reagent (colloidal gold) according to the present invention, and the results showed that the correlation r2= 0.9972 and the correlation was good (fig. 4). The detection result of the chemiluminescent detection kit is compared with the detection result of colloidal gold, and the detection result is visible: the sensitivity is higher and the linear range is wider.
6. Evaluation of sample treatment fluid stability
The obtained 33 clinical samples are stored at 2-8 ℃ and are detected at 24h, 48h, 72h and 96h respectively, and the detection results of the samples in table 6 at 2-8 ℃ are seen, the sample treatment liquid provided by the invention is used for storing the collected samples, the maximum relative deviation is-6.1% and the average deviation is-0.25% as shown in the results of 24 hours; the 48 hour results showed a maximum deviation of-7.14% and an average deviation of-1.40%; the 72 hour results showed a maximum deviation of-36.4% and an average deviation of-13.77%; 96 hours results showed a maximum deviation of-61.77% and an average deviation of-24.98%.
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In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (7)
1. A human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit comprising the following reagents:
the first reagent is a solution of streptavidin magnetic microspheres and biotinylated IGFBP-1 antibody; and
a second reagent which is an IGFBP-1 antibody labeled by alkaline phosphatase; and
sample treatment fluid;
the sample treatment fluid comprises the following components in mass fraction:
matrix buffer 0.02-0.1M/L
NaCl 0.9%
Matrix metalloproteinase inhibitor 10.0-50.0mM/mL
Brij-35 0.5%
0.05 to 0.3 percent of polyvinylpyrrolidone
Sodium dodecyl sulfate 0.01-0.025%
Trehalose 0.05% -0.5%
BSA 0.1%-0.5%
Dithiothreitol 0.005-0.03%
Sodium bisulphite 0.02-0.1%
0.02% -0.05% of chlorhexidine acetate.
2. The human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit of claim 1 wherein the streptavidin magnetic microsphere is prepared by covalently coupling an activated carboxyl magnetic microsphere to streptavidin.
3. The human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit of claim 1 wherein the concentration of the first reagent is from 50ug/mL to 300ug/mL.
4. The human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit of claim 1 wherein the concentration of the second reagent is from 0.05ug/ml to 0.200ug/ml.
5. A method of preparing a human insulin-like growth factor binding protein-1 chemiluminescent immunoassay kit of any one of claims 1-4 comprising the steps of:
s1: preparation of the first reagent:
s101: carboxyl magnetic microsphere activation: taking the suspension magnetic microsphere liquid into a centrifuge tube; removing the supernatant, adding 0.1M MES, suspending the magnetic microspheres, and repeatedly cleaning the magnetic microspheres for 3-5 times; adding EDC solution and N-hydroxysuccinimide solution to activate the magnetic microspheres, and repeatedly cleaning the magnetic microspheres for 2-5 times;
s102: covalent coupling of activated magnetic microspheres with streptavidin: preparing a reaction solution, adding activated carboxyl magnetic microspheres into the reaction solution, fully and uniformly mixing the suspension magnetic microspheres for reaction, removing supernatant in a centrifuge tube, adding 0.1M MES, suspending the magnetic microspheres, and repeatedly cleaning the magnetic microspheres for 3-5 times; adding the reaction solution into the washed magnetic microspheres, fully and uniformly mixing to suspend the magnetic microspheres for reaction, removing supernatant in a centrifuge tube, washing the magnetic microspheres with TBST solution, and repeating the washing for 3-5 times;
s103: biotinylated antibody: preparing a Biotin labeling reagent NHS-Biotin, placing the NHS-Biotin into a centrifuge tube, adding IGFBP-1 antibody purified by PBS buffer solution, fully blowing and uniformly mixing, standing at 37 ℃ in a dark place for 15min, and incubating at room temperature in a dark place for 45min in a rotating way; transferring the reacted biotinylated antibody into a dialysis bag for dialysis, and transferring into a centrifuge tube;
s104: the streptavidin magnetic microsphere is connected with a biotinylation antibody: preparing a connection reaction solution; placing streptavidin magnetic microspheres on a magnetic separation frame, and removing a supernatant; adding the connection reaction liquid, placing the connection reaction liquid on a magnetic separation frame, removing the supernatant, and repeatedly cleaning for 2-3 times; suspending streptavidin magnetic microspheres by using the connection reaction liquid, adding a biotinylation antibody, supplementing the reaction volume by using the connection reaction liquid, reacting, removing supernatant in a centrifuge tube, and repeatedly cleaning for 3-5 times;
s2: preparation of the second reagent:
s201: antibody treatment: adding the antibody with the adjusted concentration into SATA solution, performing light-proof rotary reaction at room temperature, and purifying and concentrating with PBS buffer solution for 5-8 times after the reaction is finished; collecting antibody, adding hydroxylamine hydrochloride solution, reacting at room temperature, purifying and concentrating with PBS buffer solution for 5-8 times after the reaction is finished;
s202: alkaline phosphatase activation: weighing SMCC, dissolving in DMSO, and preparing into SMCC; adding the SMCC solution into the PBS solution to obtain the SMCC solution; adding an ALP solution into an SMCC solution, and reacting at room temperature; dialyzing with PBS buffer solution after the reaction is finished;
s203: marking: collecting ALP activating enzyme after dialysis and purification, adding the treated antibody, supplementing the volume with PBS buffer solution, and reacting at room temperature in a dark place; dialyzing with PBS buffer solution after the reaction is finished; collecting the antibody after dialysis and purification, and diluting to the use concentration by using a marking buffer solution;
s3: sample treatment liquid was prepared.
6. The method for preparing a chemiluminescent immunoassay kit for human insulin-like growth factor binding protein-1 according to claim 5 wherein in step S102, the reaction solution is prepared by the following steps:
0.15mg of streptavidin is dissolved in 1.35mL of 0.1M MES solution, the pH of the MES solution is 4.0-7.0, 1-hydroxybenzotriazole 150ul dissolved in tetrahydrofuran is added, the concentration of 1-hydroxybenzotriazole is 10mg/mL, and the mixture is fully and uniformly mixed.
7. The method for preparing a chemiluminescent immunoassay kit for human insulin-like growth factor binding protein-1 according to claim 5 wherein in step S104, the method for preparing the ligation reaction solution comprises:
0.05g of polyethylene glycol and 0.09g of NaCl are weighed and dissolved in 10mL of 10mM PBS buffer solution, and the pH value of the obtained connection reaction solution is 8.0-8.5.
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| Publication number | Priority date | Publication date | Assignee | Title |
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